replication (Plant virus process)

P.N. Sharma Department of , CSK HPKV, Palampur (H.P.) INTRODUCTION  Virus infection & replication – A synonym  Virus is a nucleo- having RNA or DNA as a genetic material.

 RNA or DNA may be ss or ds, ssRNA may be +ve or –ve sense.

 Most of plant virus infect a limited number of different plant species and a few have a wide range.

do not produce any kind of reproductive structure, they multiply by using host machinery. CONCEPT

 Two separate system involved in synthesis of protein and  These two components then combine & form a nucleo-protein particle › Thus involves two inter-related biosynthetic mechanisms of the host  The n-acid directs the synthesis of progeny n-acid and this in turn, › In association with host 's form by multistep process. Host functions used by plant viruses

• Components for virus replication • (amino-acids & nucleotides synthesized by host metabolism for virus) • Energy: • involved in polymerization of viral proteins and n-RNA synthesis as nucleoside triphosphate • Protein synthesis • Viruses use , tRNA and associated enzymes & factors of host • Involve 80S cytoplasmic ribosome system • Nucleic acid synthesis • Generally almost all viruses code for an enzyme or enzymes involved in synthesis of their n-acids but may not contribute the polypeptides involved • Some viruses need host polymerases like Caulimoviruses the viral DNA enters host and transcribe into RNA form by host’s DdRp II. • In majority of RNA viruses, RdRp is involved in replication complex. • Structural components of the cell • Membrane bound complexes ( endoplasmic reticulum; cytoskeleton

REPLICATION STRATEGY • Virus absorption into host cells through wounds • Virus entry into host cells and to replication sites in the cells • Uncoating or disassembly of n-acid • and transcription of the viral : • Replication of viral n-acid • Viral genome either translated directly if +ve ssRNA or mRNAs • Gene products like replicases or viral coat protein and other proteins • Assembly or maturation of new virus particles • Transport o infective particles from cell to cell and over long distances with in the

VIRAL GENOME EXPRESSION • I: is transcription of dsDNA usually by host-DdRP • II: is transcription of ssDNA to give ds template for I (Gemini viruses) • III: is transcription of dsRNA usually by virus coded RdRp (Reoviruses) • IV: is replication of +ve stand RNA via –ve stand template by virus coded RdRp; the viral (+) strand is often the template for the early translation (=ve sense RNA viruses) • V: is the transcription o _ve sense virus genome by virus coded RdRp ( Tospoviruses) • VI: is the reverse transcription of RNA stage of retro and para- leading to dsDNA template for mRNA transcription. MAJOR EVENTS

Entry Replication Movement MODE OF ENTRY

 Wounds Mechanically TMV, BCMV, PVX, PYDV, Vectors PLRV,TLCV, WTV

 Pollen Grain Prunus necrotic ring spot, Tobacco ring spot of soybean Barley stripe

 Seeds or vegetative propagation BCMV, CMV, Pea stripe virus

 Pinocyte CPMV, Pea enation mosaic Cowpea Chloretic mottle virus

 Fungal parasite TNV , Barley yellow mosaic virus Tobacco stunt virus

 Ectodesmata TMV

 Epidermal hair TMV UNCOATING OF VIRUS PARTICLE

 Uncoating is removal of virus protein which is their around the nucleic acid.

 The ability of uncoating depends upon structure of virus coat protein. Different experiments indicating uncoating of viral RNA

 TMV multiplication detected some hours earlier when inoculation is done by RNA.

 Symptoms appears several hours earlier if naked TMV RNA is used as inoculum.

 Infectible sites produced by TMV RNA inoculum become resistant to UV radiation immediately after inoculation. MECHANISM OF REPLICATION • Production of new RNA molecule identical to template RNA is called Replication. • Viral RNA performs two function • It serves as mRNA, immediately binds to ribosome and directs the synthesis of virus specific proteins. • Among which one protein inhibits host protein and host RNA synthesis, and • other protein is specific viral replicating enzyme i.e. RNA polymerase or replicase.  Parental viral RNA is then displaced from ribosome, triggers the replication of viral RNA during which it behaves as a template or plus strand for synthesis of minus strand in presence of specific replicase already synthesized. RNA VIRUS STRATEGIES

RNA -> RNA RNA-dependent RNA polymerase RNA -> DNA RNA-dependent DNA polymerase - Host cell DNA -> RNA DNA-dependent RNA polymerase ALL RNA VIRUSES CODE FOR A POLYMERASE NEED TO MAKE MRNA

PLUS (POSITIVE) SENSE RNA

(+ve) sense mRNA

AAA NEED TO MAKE MRNA

MINUS (NEGATIVE) SENSE RNA GENOMES

(+ve) sense mRNA AAA

(-ve) sense genomic RNA NEED TO MAKE mRNA MINUS (NEGATIVE) SENSE RNA GENOMES

RNA polymerase must be packaged in virion

(+ve) sense mRNA AAA

If used, RNA modifying (-ve) sense genomic RNA enzymes are packaged in virion. NEED TO MAKE mRNA

DOUBLE-STRANDED RNA GENOMES

RNA polymerase must be packaged in virion.

(+ve) sense mRNA AAA

If used, RNA modifying Double-stranded genomic RNA enzymes are packaged in virion. NEED TO MAKE MRNA

RETROVIRUSES Reverse transcriptase must be packaged in virion. DS DS DNA DNA

+VE RNA • Once viral genome enter the cell- replication start • Its mediated by a replicase enzyme, • possibly coded partially by the viral genome and host genome. • In the presence of polymerase, a –ve stand complementary to the genome +ve strand is formed. • -ve strand serve as template for the synthesis of progeny or new +ve stand RNA by means of replicase enzyme forming a partially double stranded, partially single stranded structure, called Replicative intermediate (RI)

• A part of viral RNA form coat protein • Synthesis of new RNA is from the 3’ to 5’ ends of the templates. • Replication occurs in a replication complex that comprises of the template, newly synthesized RNA, the replicase and host factors. • The viral RNA synthesizing systems have been shown to produce two kinds of RNA structures:

• Replicative form (RF)- it is fully base paired ds and may represent RNA molecules that have seized the replication • Replication intermediate (RI) is only partly ds and contains several ss tails. This structure is closely related to that actually replicating the viral RNA.

RI: Protein Synthesis in

Infection Cycle of TMV

Sites for multiplication

 Mostly nucleus and cytoplasm serve as sites for replication and assembly of virus.

 In some cases, and other cell organelles perform the same function.

 In Potex, Caulimo, Gemini and Tobamo virus replication and assembly takes place in nucleus and from where it moves to cytoplasm.

 In Bromo, Poty, Nepo, Como viruses, replication and assembly takes place in cytoplasm.

 In Tymoviruses replication takes place in chloroplast. entry/penetration* Attachment/Penetration: bind to specific surface receptors; Entry: fuse with or engulfed by the genome plasma membrane uncoated mechanical introduction initially. NO evidence attach to specific receptor sites on mRNA replication

Transmission: : aerosols, break in proteins skin, blood, sexual contact : , fungi, , abrasion, seeds, pollen, vegetative propagation progeny virion assembly Release: lyse cells or bud through plasma membrane “channel” through “release” * wall (MPs) without lysis GENOME STRUCTURE •Seven ORFs on CaMV genome •Translation of seven proteins from two transcripts Translation regulator

Movement protein •ORF 2 is the only dispensable ORF Helper component •ORFs 6 and 7 are Inclusion, involved in translation transactivator Replication regulation factor •Packaged genomic DNA has discontinuities Reverse transcriptase Coat on both strands protein •Replication is from tRNAmet primer

CAULIMOVIRUS LIFE CYCLE

• Virus enters plant cell, protein is removed Helper Inclusion factor • dsDNA enters nucleus; gaps closed; transcription to 35S and 19S Mature particle with DNA • In the cytoplasm, the 19S RNA is translated to produce protein that forms inclusion bodies •Five ORFs are translated from 35S RNA by complex combination of strategies •Other copies of 35S RNA are reverse transcribed and packaged into virions •Particles exit cells through plasmodesmata or by Rolling Circle DNA Replication • Common in plasmid or DNA and and the circular RNA genome of viroidse.g. geminiviruses • Rolling circle DNA replication is initiated by an initiator protein encoded by the plasmid or bacteriophage DNA, which nicks one strand of the double-stranded, circular DNA molecule at a site called the double-strand origin, or DSO. • The initiator protein remains bound to the 5' phosphate end of the nicked strand, and the free 3' hydroxyl end is released to serve as a primer for DNA synthesis by DNA polymerase III. • Using the unnicked strand as a template, replication proceeds around the circular DNA molecule, displacing the nicked strand as single-stranded DNA. Displacement of the nicked strand is carried out by a host-encoded called PcrA (plasmid copy reduced) in the presence of the plasmid replication initiation protein. • Continued DNA synthesis can produce multiple single-stranded linear copies of the original DNA in a continuous head-to-tail series called a concatemer. • These linear copies can be converted to double-stranded circular molecules through the following process: • First, the initiator protein makes another nick to terminate synthesis of the first (leading) strand. RNA polymerase and DNA polymerase III then replicate the single-stranded origin (SSO) DNA to make another double-stranded circle. DNA polymerase I removes the primer, replacing it with DNA, and DNA ligase joins the ends to make another molecule of double-stranded circular DNA. • Rolling circle replication has found wide uses in academic research and , and has been successfully used for amplification of DNA from very small amounts of starting material.

Ss DNA Virus Replication

• Involves rolling circle replication; It is a two step process • 1. In the first step ss (+) strand is the template for the synthesis of (-) stand to generate a ds, replicative form (RF). This RF has two functions ie. It is the template for the transcription & is the template for the (+) strand synthesis generating free ssDNA. • The priming of the (-) strand sunthesis us usually by an RNA molecule that is generated thorugh RNA polymerase or DNA primase activity. (+) stand sunthesis is primed by a site specifiic nick in the (+) stand of the RF

• has ss (+ve)RNA genome with a VPg protein covalently linked to its 5’end and encapsulated in about 2000 monomeric units of 32-35 kDa coat protein (Flores et al.,, 2003; Betty et al., 2008). • BCMV encodes 8 proteins whose functions have been characterized (Bos 1971, Urcuqui-Inchima et al., 2001). • The coding ORF is translated into one polyprotein of 340-370 Da which is processed into: • proteinase (Pro-1), • helper component proteinase (Hc-Pro), • proteinase (Pro-3), • a 6 kDa protein, • cylindrical inclusion (CI), • a second 6 kDa protein product, • nuclear inclusion a (NIa), • nuclear inclusion b (NIb) and • cp (Flores et al., 2003). A consensus potyvirus genome map MOVEMENT OF PLANT VIRUSES Movement of Plant Viruses Short distance Long distance movement movement

Cell to Cell movement

Movement of plant viruses as Movement of plant nucleoprotein e.g. viruses as virion e.g. cucumo, bromo, tospo, nepo, como, tobamo, alfalfa caulimo viruses viruses group MOVEMENT OF VIRUS AS NUCLEOPROTEIN

Virus particle encode MP

MP binds to viral RNA

Binding unfolds viral RNA from random coil into linear rod shape structure

MP RNA complex targeted to PD

Through PD, movement to other cells

MOVEMENT OF VIRUS AS VRION

(Intercellular movement)

Formation of tubular structure in association with or through PD

Virus particle in linear form are observed inside these structure

Through cell wall, tubular structure move into neighbouring cells.

Deliver virus into that cell Cell-to-cell Movement

Infected Cell cell wall Healthy Cell plasmamembrane

spikes Desmotubule =>ER sleeve central cavity neck

Virus Movement Proteins (MPs) enable viral movement through plasmodesmata Cell-to-cell movement strategies

Classification

TMV-like Potyvirus-like Closterovirus Double Gene Block Triple Gene Block Geminivirus Viroids Tubule formers Different strategies can complement

Example: AMV ( ) Needs MP plus CP

AMV MP can be complemented by: 1. CPMV MP (tubules, virion) 2. CMV MP (CP necessary) 3. TMV MP (no CP necessary)

Sanchez-Navarro et al (2006) 346:66 Different strategies

MP-RNA MP-RNA-CP virions

CP-RNA or virions Extreme comparison

(TMV) (TSWV)

TMV, +RNA, no envelope CP not required RNP passes through PD

TSWV, +/-RNA, enveloped CP (N) protein required RNP in tubules that grow through PD

TSWV and TMV represent two extremes, or do they? Transport of virus replication complexes (VRCs)

TMV directed transport of VRC to and perhaps through PD

directed transport of VRC via tubules

TSWV Complementation of TMV MP by TSWV MP

TMVDMP RNA TSWV MP moves TMV

Tubules are formed TSWV-MP

Lewandowski and Adkins (2005) Virology 342:26 Long distance Movement Within Plant

Resistant, Immune, . e . i host . susceptible and Tolerant

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CONCLUSION

ACKNOWLEDGEMENTS

• I gratefully acknowledge the use of text book “Matthews Plant Virology” by Roger Hull. • I acknowledge the scientists who spent valuable time in generating information on various aspects of plant Virology and displayed the same on internet for use by students, teachers and researchers PN Sharma